Polysaccharide from aerial part of Chuanminshen violaceum alleviates oxidative stress and inflammatory response in aging mice through modulating intestinal microbiota

Abstract

Aging is a biological process of progressive deterioration of physiological functions, which poses a serious threat to individual health and a heavy burden on public health systems. As population aging continues, research into anti-aging drugs that prolong life and improve health is of particular importance. In this study, the polysaccharide from stems and leaves of Chuanminshen violaceum was obtained with water extraction and alcohol precipitation, and then separated and purified with DEAE anion exchange chromatography and gel filtration to obtain CVP-AP-I. We gavaged natural aging mice with CVP-AP-I and performed serum biochemical analysis, histological staining, quantitative real-time PCR (qRT-PCR) and ELISA kit assays to analyze inflammation and oxidative stress-related gene and protein expression in tissues, and 16SrRNA to analyze intestinal flora. We found that CVP-AP-I significantly improved oxidative stress and inflammatory responses of the intestine and liver, restored the intestinal immune barrier, and balanced the dysbiosis of intestinal flora. In addition, we revealed the potential mechanism behind CVP-AP-I to improve intestinal and liver function by regulating intestinal flora balance and repairing the intestinal immune barrier to regulate the intestinal-liver axis. Our results indicated that C. violaceum polysaccharides possessed favorable antioxidant, anti-inflammatory and potentially anti-aging effects in vivo.

Keywords: Chuanminshen violaceum; antiinflammatory; antioxidation; gut microbiota; gut-liver axis; polysaccharide.

Figure 1

Schematic overview of experimental design.

Figure 2

Isolation, chemical composition, monosaccharide composition and average-molecular weight of CVSS polysaccharides. (A) The elution curve of CVP-AP on DEAE anion exchange chromatography. A single component is obtained. (B) CVP-AP-I elution profile was obtained after gel filtration purification. (C) The chemical compositions of CVP-AP-I including monosaccharide composition (mol %), total carbohydrate (%), protein content (%), polyphenol content (%). (D) The molecular weight determination of CVSS polysaccharide by gel permeation chromatography.

Figure 3

Effects of CVP-AP-I on body weight and serum levels of ALT, AST, pro-inflammatory factors (IL-6, IL-1β, TNF-α), antioxidant enzymes (SOD, CAT, GPX) and MDA in aging mice. (A) Changes in body weight of mice (Relative to initial body weight; n=10); (B) Quantification shows the serum levels of ALT and AST in the mice of different groups (n=4); (C) Quantification shows the inflammatory factor IL-1, TNF-α, IL-6 level in the serum from different groups mice(n=6). (D) Quantification shows the activity of the antioxidant enzymes SOD, CAT and GPX and the level of MDA in the serum of the mice in the different groups (n=6). All data are represented as means ± SD.*p<0.05, **p<0.01, and ***p<0.001 as compared with Control Group. ##p<0.01 and ###p<0.001 as compared with CVP-AP-I (L) group.

Figure 4

Effects of CVP-AP-I on antioxidant and anti-inflammatory capacity of intestine and liver in naturally aging mice. (A) qRT-PCR results show the relative expression levels of inflammatory factors IL-1β, TNF-α and IL-6 genes in liver and intestinal tissues (duodenum, jejunum and ileum) of different groups of mice (n=5); (B) qRT-PCR results show the relative expression levels of the antioxidant enzymes SOD, CAT and GPX genes in liver and intestinal tissues (duodenum, jejunum and ileum) of different groups of mice (n=5); (C) qRT-PCR results show the relative expression levels of transcription factor Nrf2 gene in liver and intestinal tissues (duodenum, jejunum and ileum) of different groups of mice (n=5); (D) Quantitative results show the protein expression levels of antioxidant enzymes SOD, CAT, GPX and T-AOC in jejunum and liver of mice in different groups (n=6); (E) Quantitative results show the protein expression levels of inflammatory factors IL-1β, TNF and IL-6 in jejunum and liver of mice in different groups (n=6); (F) Quantitative results show the protein expression of ROS and MDA in jejunum and liver of mice in each group (n=6); All data are represented as means ± SD. *p<0.05, **p<0.01, and ***p<0.001 as compared with Control Group. #p<0.05 and ##p<0.01 as compared with CVP-AP-I (L) group.

Figure 5

Effects of CVP-AP-I on liver and intestinal histology and intestinal immune barrier in naturally aging mice. (A) Representative images of H.E staining show that CVP-AP-I improves age-induced liver lesions. (“→” Inflammatory cell necrosis of the liver; “→” Multinucleated hepatocytes; “→” Mild steatosis of hepatocytes); (B-D) Representative images of H.E staining show that CVP-AP-I reverses aging-induced villus and crypt defects in the ileum, jejunum and duodenum; (E) Quantification shows the levels of TG and TC in the serum of different groups of mice(n=4); (F-H) Quantitative show of CVP reversal of aging-induced intestinal villus and crypt defects in the ileum, jejunum and duodenum. Error bars indicate Mix to Max (n = 6); (I) qRT-PCR results show the expressions of Occludin, ZO-1 and Muc2 in duodenum, jejunum and ileum of mice from different groups (n=4); (J) Quantification shows LPS levels in the serum and liver of different groups of mice (n=4); (K) Quantification shows sIgA levels in the jejunum of different groups of mice (n=6). All data are expressed as mean ± SD. *p<0.05, **p<0.01, and ***p<0.001 as compared with Control Group. #p<0.05, ##p<0.01, and ###p<0.001 as compared with CVP-AP-I(L) group.

Figure 6

CVSS polysaccharides modulate the composition and structure of gut microflora. (A) OUT Rarefaction Curves of gut microbiota in different groups; (B) Venn diagram showing the unique and shared OTUs from different groups; (C) Bacterial community richness measured by Chao1 index in different groups; (D) Bacterial community diversity measured by Shannon index in different group; (E) Unweighted Unifrac Principal Coordinate Analysis by bacterial microbiota; (F) NMDS ordination based on Bray-Curtis similarities of bacterial communities; (G) UPGMA clustering tree based on unweighted Unifrac distances. “*” stands for the comparation with Control; **p<0.01 by one-way ANOVA.

Figure 7

Comparative analysis of the effects of CVSS polysaccharide supplementation on the gut microflora. (A-C) Relative abundance of species in the top 10 of the intestinal flora at the phylum, genus and species level; (D) Quantification shows the abundance ratio of Firmicutes/Bacteroidetes at the phylum level, n=5, “*” stands for the comparation with Control; *p<0.05 by one-way ANOVA; (E) The histogram of the distribution of LDA values shows the species with significant differences in abundance in different groups; (F) Heatmap depicting the relative abundance of 20 bacterial species significantly enriched in different samples at the genus level.

Figure 8

Heatmap showing the correlation of gut microbial genera with pro-inflammatory factors (IL-6, IL-1β, TNF-α), antioxidant enzymes (CAT, SOD, GPX), MDA, LPS, sIgA; (A) indicates correlation with serum; (B) indicates correlation with jejunum; (C) indicates correlation with liver. The colour and shape of the ellipse correlates with the strength of the correlation, with darker and flatter ellipses being more strongly correlated (*p<0.05, **p<0.01, ***p<0.001).

Figure 9

Summary diagram of the pharmacological effects of CVP-AP-I.